The lack of Tex44 causes severe subfertility with flagellar abnormalities in male mice.

By analyzing a mouse Interspecific Recombinant Congenic Strain (IRCS), we previously identified a quantitative trait locus (QTL), called Mafq1 on mouse chromosome 1, that is associated with male hypofertility and ultrastructural sperm abnormalities. Within this locus, we identified a new candidate gene that could be implicated in a reproductive phenotype: Tex44 (Testis-expressed protein 44). We thus performed a CRISPR/Cas9-mediated complete deletion of this gene in mice in order to study its function. Tex44-KO males were severely hypofertile in vivo and in vitro due to a drastic reduction of sperm motility which itself resulted from important morphological sperm abnormalities. Namely, Tex44-KO sperm showed a disorganized junction between the midpiece and the principal piece of the flagellum, leading to a 180° flagellar bending in this region. In addition, the loss of some axonemal microtubule doublets and outer dense fibers in the flagellum's principal piece has been observed. Our results suggest that, in mice, TEX44 is implicated in the correct set-up of the sperm flagellum during spermiogenesis and its absence leads to flagellar abnormalities and consequently to severe male hypofertility.

Decreased embryo developmental potential and lower cumulative pregnancy rate in men with multiple morphological abnormalities of the sperm flagella.

Objective: Multiple morphological abnormalities of the sperm flagella (MMAF) is characterized by abnormal flagellar phenotypes, which is a particular kind of asthenoteratozoospermia. Previous studies have reported a comparable intracytoplasmic sperm injection (ICSI) outcome in terms of fertilization rate and clinical pregnancy rate in patients with MMAF compared with those with no MMAF; however, others have conflicting opinions. Assisted reproductive technology (ART) outcomes in individuals with MMAF are still controversial and open to debate. Methods: A total of 38 patients with MMAF treated at an academic reproductive center between January 2014 and July 2022 were evaluated in the current retrospective cohort study and followed up until January 2023. Propensity score matching was used to adjust for the baseline clinical characteristics of the patients and to create a comparable control group. The genetic pathogenesis of MMAF was confirmed by whole exome sequencing. The main outcomes were the embryo developmental potential, the cumulative pregnancy rate (CLPR), and the cumulative live birth rate (CLBR). Results: Pathogenic variants in known genes of DNAH1, DNAH11, CFAP43, FSIP2, and SPEF2 were identified in patients with MMAF. Laboratory outcomes, including the fertilization rate, 2PN cleavage rate, blastocyst formation rate, and available blastocyst rate, followed a trend of decline in the MMAF group (p < 0.05). Moreover, according to the embryo transfer times and complete cycles, the CLPR in the cohort of MMAF was lower compared with the oligoasthenospermia pool (p = 0.033 and p = 0.020, respectively), while no statistical differences were observed in the neonatal outcomes. Conclusion: The current study presented decreased embryo developmental potential and compromised clinical outcomes in the MMAF cohort. These findings may provide clinicians with evidence to support genetic counseling and clinical guidance in specific patients with MMAF.

Genetic mutation of Cep76 results in male infertility due to abnormal sperm tail composition.

The transition zone is a specialised gate at the base of cilia/flagella, which separates the ciliary compartment from the cytoplasm and strictly regulates protein entry. We identified a potential new regulator of the male germ cell transition zone, CEP76. We demonstrated that CEP76 was involved in the selective entry and incorporation of key proteins required for sperm function and fertility into the ciliary compartment and ultimately the sperm tail. In the mutant, sperm tails were shorter and immotile as a consequence of deficits in essential sperm motility proteins including DNAH2 and AKAP4, which accumulated at the sperm neck in the mutant. Severe annulus, fibrous sheath, and outer dense fibre abnormalities were also detected in sperm lacking CEP76. Finally, we identified that CEP76 dictates annulus positioning and structure. This study suggests CEP76 as a male germ cell transition zone protein and adds further evidence to the hypothesis that the spermatid transition zone and annulus are part of the same functional structure.

A novel homozygous nonsense variant of AK7 is associated with multiple morphological abnormalities of the sperm flagella.

RESEARCH QUESTION: Is the novel homozygous nonsense variant of AK7 associated with multiple morphological abnormalities of the sperm flagella (MMAF), a specific type of oligoasthenoteratozoospermia leading to male infertility? DESIGN: Whole-exome sequencing and Sanger sequencing were performed to identify potential gene variants. Immunoblotting and immunofluorescence were applied to confirm the relationship between mutated genes and disease phenotypes. The concentration of reactive oxygen species and the rate of apoptosis were measured to evaluate the mitochondrial function of spermatozoa. Transmission electron microscopy and scanning electron microscopy were employed to observe sperm ultrastructure. RESULTS: A novel homozygous nonsense variant of AK7, c.1153A>T (p. Lys385*), was identified in two infertile siblings with asthenoteratozoospermia through whole-exome sequencing. Both immunoblotting and immunofluorescence assays showed practically complete absence of AK7 in the patient's spermatozoa. Additionally, the individual with the novel AK7 variant exhibited a phenotype characterized by severe oxidative stress and apoptosis caused by mitochondrial metabolic dysfunction of spermatozoa. Notably, remarkable flagellar defects with multiple axonemes in uniflagellate spermatozoa, accompanied by mitochondrial vacuolization, were observed; this has not been reported previously in patients with other AK7 variants. CONCLUSIONS: This study found that a novel identified homozygous nonsense variant of AK7 may be associated with MMAF-related asthenoteratozoospermia. The observed functional associations between mitochondria and sperm flagellar assembly provide evidence for potential mutual regulation between AK7 and flagella-associated proteins during spermatogenesis.

Flagellar pH homeostasis mediated by Na+/H+ exchangers regulates human sperm functions through coupling with CatSper and KSper activation.

STUDY QUESTION: Whether and how do Na+/H+ exchangers (NHEs) regulate the physiological functions of human sperm? SUMMARY ANSWER: NHE-mediated flagellar intracellular pH (pHi) homeostasis facilitates the activation of the pH-sensitive, sperm-specific Ca2+ channel (CatSper) and the sperm-specific K+ channel (KSper), which subsequently modulate sperm motility, hyperactivation, flagellar tyrosine phosphorylation, and the progesterone (P4)-induced acrosome reaction. WHAT IS KNOWN ALREADY: Sperm pHi alkalization is an essential prerequisite for the acquisition of sperm-fertilizing capacity. Different sperm functions are strictly controlled by particular pHi regulatory mechanisms. NHEs are suggested to modulate sperm H+ efflux. STUDY DESIGN, SIZE, DURATION: This was a laboratory study that used samples from >50 sperm donors over a period of 1 year. To evaluate NHE action on human sperm function, 5-(N,N-dimethyl)-amiloride (DMA), a highly selective inhibitor of NHEs, was utilized. All experiments were repeated at least five times using different individual sperm samples or cells. PARTICIPANTS/MATERIALS, SETTING, METHODS: By utilizing the pH fluorescent indicator pHrodo Red-AM, we detected alterations in single-cell pHi value in human sperm. The currents of CatSper and KSper in human sperm were recorded by the whole-cell patch-clamp technique. Changes in population and single-cell Ca2+ concentrations ([Ca2+]i) of human sperm loaded with Fluo 4-AM were measured. Membrane potential (Vm) and population pHi were quantitatively examined by a multimode plate reader after sperm were loaded with 3,3'-dipropylthiadicarbocyanine iodide and 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein, acetoxymethyl ester, respectively. Sperm motility parameters were assessed by a computer-assisted semen analysis system. Tyrosine phosphorylation was determined by immunofluorescence, and sperm acrosome reaction was evaluated by Pisum sativum agglutinin-FITC staining. MAIN RESULTS AND THE ROLE OF CHANCE: DMA-induced NHEs inhibition severely acidified the human sperm flagellar pHi from 7.20 ± 0.04 to 6.38 ± 0.12 (mean ± SEM), while the effect of DMA on acrosomal pHi was less obvious (from 5.90 ± 0.13 to 5.57 ± 0.12, mean ± SEM). The whole-cell patch-clamp recordings revealed that NHE inhibition remarkably suppressed alkalization-induced activation of CatSper and KSper. As a consequence, impairment of [Ca2+]i homeostasis and Vm maintenance were detected in the presence of DMA. During the capacitation process, pre-treatment with DMA for 2 h potently decreased sperm pHi, which in turn decreased sperm motility and kinetic parameters. Sperm capacitation-associated functions, including hyperactivation, tyrosine phosphorylation, and P4-induced acrosome reaction, were also compromised by NHE inhibition. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: This was an in vitro study. Caution should be taken when extrapolating these results to in vivo applications. WIDER IMPLICATIONS OF THE FINDINGS: This study revealed that NHEs are important physiological regulators for human CatSper and KSper, which are indispensable for human sperm fertility, suggesting that malfunction of NHEs could be an underlying mechanism for the pathogenesis of male infertility. FUNDING/COMPETING INTEREST(S): This work was supported by the National Natural Science Foundation of China (32271167 and 81871202 to X.Z.), Jiangsu Innovation and Entrepreneurship Talent Plan (JSSCRC20211543 to X.Z.), the Social Development Project of Jiangsu Province (No. BE2022765 to X.Z.), the Society and livelihood Project of Nantong City (No. MS22022087 to X.Z.), and the Natural Science Foundation of Jiangsu Province (BK20220608 to H.K.). The authors have no competing interests to declare.

Coiled-coil domain containing 159 is required for spermatid head and tail assembly in mice†.

The centrosome is critical for maintaining the sperm head-tail connection and the formation of flagellar microtubules. In this study, we found that in mouse testes, CCDC159 (coiled-coil domain-containing protein 159) is specifically localized to the head-tail coupling apparatus (HTCA) of spermatids, a structure that ensures sperm head-tail tight conjunction. CCDC159 contains a C-terminal coiled-coil domain that functions as the centrosomal localization signal. Gene knockout (KO) of Ccdc159 in mice resulted in acephalic spermatozoa, abnormal flagella, and male infertility. To explore the mechanism behind CCDC159 regulating spermatogenesis, we identified CCDC159-binding proteins using a yeast two-hybrid screen and speculated that CCDC159 participates in HTCA assembly by regulating protein phosphatase PP1 activity. Further RNA-sequencing analyses of Ccdc159 KO testes revealed numerous genes involved in male gamete generation that were downregulated. Together, our results show that CCDC159 in spermatids is a novel centrosomal protein anchoring the sperm head to the tail. Considering the limitation of KO mouse model in clarifying the biological function of CCDC159 in spermatogenesis, a gene-rescue experiment will be performed in the future.

Morphological analysis of the human sperm tail during the early post-fertilization stage.

After fertilization, the human sperm tail lost its fibrous sheath at the pronuclear stage to become coiled and then attached to one of the first mitotic spindle poles. The tip of the sperm tail was branched and its degree did not change from the pronuclear stage to the second mitosis.

The Cby3/ciBAR1 complex positions the annulus along the sperm flagellum during spermiogenesis.

Proper compartmentalization of the sperm flagellum is essential for fertility. The annulus is a septin-based ring that demarcates the midpiece (MP) and the principal piece (PP). It is assembled at the flagellar base, migrates caudally, and halts upon arriving at the PP. However, the mechanisms governing annulus positioning remain unknown. We report that a Chibby3 (Cby3)/Cby1-interacting BAR domain-containing 1 (ciBAR1) complex is required for this process. Ablation of either gene in mice results in male fertility defects, caused by kinked sperm flagella with the annulus mispositioned in the PP. Cby3 and ciBAR1 interact and colocalize to the annulus near the curved membrane invagination at the flagellar pocket. In the absence of Cby3, periannular membranes appear to be deformed, allowing the annulus to migrate over the fibrous sheath into the PP. Collectively, our results suggest that the Cby3/ciBAR1 complex regulates local membrane properties to position the annulus at the MP/PP junction.

A splice donor variant of GAS8 induces structural disorganization of the axoneme in sperm flagella and leads to nonsyndromic male infertility.

Motile cilia and flagella are closely related organelles structured around a highly conserved axoneme whose formation and maintenance involve proteins from hundreds of genes. Defects in many of these genes have been described to induce primary ciliary dyskinesia (PCD) mainly characterized by chronic respiratory infections, situs inversus and/or infertility. In men, cilia/flagella-related infertility is usually caused by asthenozoospermia due to multiple morphological abnormalities of the sperm flagella (MMAF). Here, we investigated a cohort of 196 infertile men displaying a typical MMAF phenotype without any other PCD symptoms. Analysis of WES data identified a single case carrying a deleterious homozygous GAS8 variant altering a splice donor consensus site. This gene, also known as DRC4, encodes a subunit of the Nexin-Dynein Regulatory Complex (N-DRC), and has been already associated to male infertility and mild PCD. Confirming the deleterious effect of the candidate variant, GAS8 staining by immunofluorescence did not evidence any signal from the patient's spermatozoa whereas a strong signal was present along the whole flagella length in control cells. Concordant with its role in the N-DRC, transmission electron microscopy evidenced peripheral microtubule doublets misalignments. We confirm here the importance of GAS8 in the N-DRC and observed that its absence induces a typical MMAF phenotype not necessarily accompanied by other PCD symptoms.

CCDC65, encoding a component of the axonemal Nexin-Dynein regulatory complex, is required for sperm flagellum structure in humans.

Sperm flagella share an evolutionary conserved microtubule-based structure with motile cilia expressed at the surface of several cell types, such as the airways epithelial cells. As a result, male infertility can be observed as an isolated condition or a syndromic trait, illustrated by Primary Cilia Dyskinesia (PCD). We report two unrelated patients showing multiple morphological abnormalities of the sperm flagella (MMAF) and carrying distinct homozygous truncating variants in the PCD-associated gene CCDC65. We characterized one of the identified variants (c.1208del; p.Asn403Ilefs*9), which induces the near absence of CCDC65 protein in patient sperm. In Chlamydomonas, CCDC65 ortholog (DRC2, FAP250) is a component of the Nexin-Dynein Regulatory complex (N-DRC), which interconnects microtubule doublets and coordinates dynein arms activity. In sperm cells from the patient, we also show the loss of GAS8, another component of the N-DRC, supporting a structural/functional link between the two proteins. Our work indicates that, similarly to ciliary axoneme, CCDC65 is required for sperm flagellum structure. Importantly, our work provides first evidence that mutations in the PCD-associated gene CCDC65 also cause asthenozoospermia.

Novel biallelic variants in DNAH1 cause multiple morphological abnormalities of sperm flagella with favorable outcomes of fertility after ICSI in Han Chinese males.

BACKGROUND: Multiple morphological abnormalities of sperm flagella is an idiopathic asthenoteratozoospermia characterized by absent, short, coiled, angulation, and irregular-caliber flagella. Genetic variants of DNAH1 gene have been identified as a causative factor of multiple morphological abnormalities of sperm flagella and intracytoplasmic sperm injection is an available strategy for infertile males with dynein axonemal heavy chain 1 defects to conceive. OBJECTIVES: To identify novel variants and candidate mutant hotspots of DNAH1 gene related to multiple morphological abnormalities of sperm flagella and male infertility in humans. MATERIALS AND METHODS: The DNAH1 variants were identified by whole exome sequencing and confirmed with Sanger sequencing. Papanicolaou staining, scanning and transmission electron microscopy, and immunostaining were performed to investigate the morphological and ultrastructural characteristics of spermatozoa. Intracytoplasmic sperm injection was applied for the assisted reproductive therapy of males harboring biallelic DNAH1 variants. RESULTS: We identified 18 different DNAH1 variants in 11 unrelated families, including nine missense variants (p.A2564T, p.T3657R, p.G1862R, p.L2296P, p.T4041I, p.L611P, p.A913D, p.R1932Q, p.R2356W) and nine loss-of-function variants (c.2301-1G>T, p.Q1518*, p.R1702*, p.D2845Mfs*2, p.P3909Rfs*33, p.Q4040Dfs*33, p.Q4058*, p.E4060Pfs*61, p.V4071Cfs*54). A total of 66.7% (12/18) of the identified variants were novel. Morphological analysis based on Papanicolaou staining and scanning electron microscopy demonstrated the typical multiple morphological abnormalities of sperm flagella characteristics of dynein axonemal heavy chain 1-deficient spermatozoa. Immunostaining further revealed the absence of inner dynein arms but not outer dynein arms, which induced a general ultrastructural disorganization, such as the loss of central pair and mis-localization of the microtubule doublets and outer dense fibers. To date, seven affected couples have accepted the intracytoplasmic sperm injection treatment, and three of them have given birth to five healthy babies. DISCUSSION AND CONCLUSION: These findings further expand the variant spectrum of DNAH1 gene related to multiple morphological abnormalities of sperm flagella and male infertility in humans, thus providing new information for the molecular diagnosis of asthenoteratozoospermia. The favorable fertility outcomes of intracytoplasmic sperm injection will facilitate the genetic counseling and clinical treatment of infertile males with multiple morphological abnormalities of sperm flagella in the future.

A novel NPHP4 homozygous missense variant identified in infertile brothers with multiple morphological abnormalities of the sperm flagella.

PURPOSE: Asthenozoospermia is an important cause of male infertility, and the most serious type is characterized by multiple morphological abnormalities of the sperm flagella (MMAF). However, the precise etiology of MMAF remains unknown. In the current study, we recruited a consanguineous Pakistani family with two infertile brothers suffering from primary infertility due to MMAF without obvious signs of PCD. METHODS: We performed whole-exome sequencing on DNAs of the patients, their parents, and a fertile brother and identified the homozygous missense variant (c.1490C > G (p.P497R) in NPHP4 as the candidate mutation for male infertility in this family. RESULTS: Sanger sequencing confirmed that this mutation recessively co-segregated with the MMAF in this family. In silico analysis revealed that the mutation site is conserved across different species, and the identified mutation also causes abnormalities in the structure and hydrophobic interactions of the NPHP4 protein. Different bioinformatics tools predict that NPHP4p.P497R mutation is pathogenic. Furthermore, Papanicolaou staining and scanning electron microscopy of sperm revealed that affected individuals displayed typical MMAF phenotype with a high percentage of coiled, bent, short, absent, and/or irregular flagella. Transmission electron microscopy images of the patient's spermatozoa revealed significant anomalies in the sperm flagella with the absence of a central pair of microtubules (9 + 0) in every section scored. CONCLUSIONS: Taken together, these results show that the homozygous missense mutation in NPHP4 is associated with MMAF.

LRRC23 truncation impairs radial spoke 3 head assembly and sperm motility underlying male infertility.

Radial spokes (RS) are T-shaped multiprotein complexes on the axonemal microtubules. Repeated RS1, RS2, and RS3 couple the central pair to modulate ciliary and flagellar motility. Despite the cell type specificity of RS3 substructures, their molecular components remain largely unknown. Here, we report that a leucine-rich repeat-containing protein, LRRC23, is an RS3 head component essential for its head assembly and flagellar motility in mammalian spermatozoa. From infertile male patients with defective sperm motility, we identified a splice site variant of LRRC23. A mutant mouse model mimicking this variant produces a truncated LRRC23 at the C-terminus that fails to localize to the sperm tail, causing male infertility due to defective sperm motility. LRRC23 was previously proposed to be an ortholog of the RS stalk protein RSP15. However, we found that purified recombinant LRRC23 interacts with an RS head protein RSPH9, which is abolished by the C-terminal truncation. Evolutionary and structural comparison also shows that LRRC34, not LRRC23, is the RSP15 ortholog. Cryo-electron tomography clearly revealed that the absence of the RS3 head and the sperm-specific RS2-RS3 bridge structure in LRRC23 mutant spermatozoa. Our study provides new insights into the structure and function of RS3 in mammalian spermatozoa and the molecular pathogenicity of LRRC23 underlying reduced sperm motility in infertile human males.

CCDC146 is required for sperm flagellum biogenesis and male fertility in mice.

Multiple morphological abnormalities of the flagella (MMAF) is a severe disease of male infertility, while the pathogenetic mechanisms of MMAF are still incompletely understood. Previously, we found that the deficiency of Ccdc38 might be associated with MMAF. To understand the underlying mechanism of this disease, we identified the potential partner of this protein and found that the coiled-coil domain containing 146 (CCDC146) can interact with CCDC38. It is predominantly expressed in the testes, and the knockout of this gene resulted in complete infertility in male mice but not in females. The knockout of Ccdc146 impaired spermiogenesis, mainly due to flagellum and manchette organization defects, finally led to MMAF-like phenotype. Furthermore, we demonstrated that CCDC146 could interact with both CCDC38 and CCDC42. It also interacts with intraflagellar transport (IFT) complexes IFT88 and IFT20. The knockout of this gene led to the decrease of ODF2, IFT88, and IFT20 protein levels, but did not affect CCDC38, CCDC42, or ODF1 expression. Additionally, we predicted and validated the detailed interactions between CCDC146 and CCDC38 or CCDC42, and built the interaction models at the atomic level. Our results suggest that the testis predominantly expressed gene Ccdc146 is essential for sperm flagellum biogenesis and male fertility, and its mutations might be associated with MMAF in some patients.

Identification of CFAP52 as a novel diagnostic target of male infertility with defects of sperm head-tail connection and flagella development.

Male infertility is a worldwide population health concern. Asthenoteratozoospermia is a common cause of male infertility, but its etiology remains incompletely understood. No evidence indicates the relevance of CFAP52 mutations to human male infertility. Our whole-exome sequencing identified compound heterozygous mutations in CFAP52 recessively cosegregating with male infertility status in a non-consanguineous Chinese family. Spermatozoa of CFAP52-mutant patient mainly exhibited abnormal head-tail connection and deformed flagella. Cfap52-knockout mice resembled the human infertile phenotype, showing a mixed acephalic spermatozoa syndrome (ASS) and multiple morphological abnormalities of the sperm flagella (MMAF) phenotype. The ultrastructural analyses further revealed a failure of connecting piece formation and a serious disorder of '9+2' axoneme structure. CFAP52 interacts with a head-tail coupling regulator SPATA6 and is essential for its stability. Expression of microtubule inner proteins and radial spoke proteins were reduced after the CFAP52 deficiency. Moreover, CFAP52-associated male infertility in humans and mice could be overcome by intracytoplasmic sperm injection (ICSI). The study reveals a prominent role for CFAP52 in sperm development, suggesting that CFAP52 might be a novel diagnostic target for male infertility with defects of sperm head-tail connection and flagella development.

Age-Related Changes in Sperm Morphology and Analysis of Multiple Sperm Defects.

BACKGROUND: Analysis of sperm morphology defects (amorphous heads, abnormal acrosome, etc.) is useful for estimating the efficiency of spermiogenesis and sperm maturation. An advanced paternal age (more than 40 years) is associated with decreasing sperm count and reduced motility; however, there is little information on the effect of aging relating to sperm morphological defects. Moreover, searching for stable combinations of certain morphological defects in the same sperm can be useful for better understanding spermiogenesis. The aim of the study was to investigate age-related changes in sperm morphology and the prevalence of certain combinations of sperm morphological defects in men from the general population. METHODS: Sperm morphology was assessed in 1266 volunteers from the Russian urban general population in different age groups (18-19, 20-24, 25-29, 30-34, 35-40, and over 40 years old). Two hundred sperm were evaluated from each semen sample (about 250 thousand spermatozoa in total). Sperm defects were classified according to the WHO laboratory manual (WHO, 2010). The total percentage of each sperm defect and the frequency of different combinations of sperm morphological anomalies for each age group were counted. Additionally, a similar analysis was performed for the groups of normospermia and pathozoospermia. RESULTS: The frequency of coiled and short sperm tails increased in men over 40 years old compared to younger subjects; however, aging did not affect the percentage of morphologically normal sperm. It was shown that the combination of a misshaped head (amorphous, pyriform, and elongated) with a postacrosomal vacuole, acrosome defect, excess residual cytoplasm, or any anomaly of the midpiece or tail in the same spermatozoon were not random combinations of independent solitary defects. The increased frequency of combinations of coiled tails with amorphous, elongated, or vacuolated heads was observed in men older than 40 years. Sperm morphological defects, such as severely deformed heads (pyriform, elongated, and round) were more common in men with pathozoospermia compared to normospermic subjects. CONCLUSIONS: An age-related impairment in sperm morphology was found. Stable combinations of head defects with anomalies in the acrosome, midpiece or tail suggest that these defects may be the result of a general violation in the morphogenetic mechanism.

Human sperm rotate with a conserved direction during free swimming in four dimensions.

Head rotation in human spermatozoa is essential for different swimming modes and fertilisation, as it links the molecular workings of the flagellar beat with sperm motion in three-dimensional (3D) space over time. Determining the direction of head rotation has been hindered by the symmetry and translucent nature of the sperm head, and by the fast 3D motion driven by the helical flagellar beat. Analysis has been mostly restricted to two-dimensional (2D) single focal plane image analysis, which enables tracking of head centre position but not tracking of head rotation. Despite the conserved helical beating of the human sperm flagellum, human sperm head rotation has been reported to be uni- or bi-directional, and even to intermittently change direction in a given cell. Here, we directly measure the head rotation of freely swimming human sperm using multi-plane 4D (3D+t) microscopy and show that: (1) 2D microscopy is unable to distinguish head rotation direction in human spermatozoa; (2) head rotation direction in non-capacitating and capacitating solutions, for both aqueous and viscous media, is counterclockwise (CCW), as seen from head to tail, in all rotating spermatozoa, regardless of the experimental conditions; and (3) head rotation is suppressed in 36% of spermatozoa swimming in non-capacitating viscous medium, although CCW rotation is recovered after incubation in capacitating conditions within the same viscous medium, possibly unveiling an unexplored aspect of the essential need of capacitation for fertilisation. Our observations show that the CCW head rotation in human sperm is conserved. It constitutes a robust and persistent helical driving mechanism that influences sperm navigation in 3D space over time, and thus is of critical importance in cell motility, propulsion of flagellated microorganisms, sperm motility assessments, human reproduction research, and self-organisation of flagellar beating patterns and swimming in 3D space.

A CUG-initiated CATSPERθ functions in the CatSper channel assembly and serves as a checkpoint for flagellar trafficking.

Calcium signaling is critical for successful fertilization. In spermatozoa, calcium influx into the sperm flagella mediated by the sperm-specific CatSper calcium channel is necessary for hyperactivated motility and male fertility. CatSper is a macromolecular complex and is repeatedly arranged in zigzag rows within four linear nanodomains along the sperm flagella. Here, we report that the Tmem249-encoded transmembrane (TM) domain-containing protein, CATSPERθ is essential for the CatSper channel assembly during sperm tail formation. CATSPERθ facilitates the channel assembly by serving as a scaffold for a pore-forming subunit CATSPER4. CATSPERθ is specifically localized at the interface of a CatSper dimer and can self-interact, suggesting its potential role in CatSper dimer formation. Male mice lacking CATSPERθ are infertile because the sperm lack the entire CatSper channel from sperm flagella, rendering sperm unable to hyperactivate, regardless of their normal expression in the testis. In contrast, genetic abrogation of any of the other CatSper TM subunits results in loss of CATSPERθ protein in the spermatid cells during spermatogenesis. CATSPERθ might act as a checkpoint for the properly assembled CatSper channel complex to traffic to sperm flagella. This study provides insights into the CatSper channel assembly and elucidates the physiological role of CATSPERθ in sperm motility and male fertility.